This proposal describes a novel methodology for the rapid identification of base-pair heterozygosity in human DNA, based on established techniques of enzymatic DNA amplification, dideoxy sequencing, and allele-specific probe hybridization. Potentially, all polymorphic sites in a screened region can be detected using this approach, and the method can be applied at any cloned locus. In contrast, less than 15% of these polymorphic sites are detectable using existing restriction enzyme-based techniques. The first major objective is to use this strategy to identify multiple polymorphic genetic markers at the human retinoblastoma locus, and to use these markers for probe-based diagnosis and genetic risk assessment in retinoblastoma-prone families. Included are some families in which existing restriction fragment length polymorphisms are uninformative, and hence molecular diagnosis of carrier status is not currently possible. Identification of additional polymorphic markers is also important in that it will reduce the potential for misdiagnosis resulting from intragenic recombination in this large (greater than 180kb), gene, a significant risk when diagnosis is based on a single marker. A second major objective is to investigate the genetic mechanisms that lead to somatic expression of the recessive tumor- predisposing allele in carriers. Previous studies in an in vitro system suggest that allele loss, often extending over large chromosomal regions, is the predominant mechanism for expression of such a recessive gene at a heterozygous locus. This will be studied at two analogous genetic loci, one under naturally occurring conditions in vivo (the retinoblastoma locus, studied in tumors), the second an artificially heterozygous locus in a human cell line studied in vitro. In both systems, multiple linked polymorphic markers within and flanking the loci will be identified, and segregation of these markers will be measured to determine the frequency, extent, and molecular nature of these allele loss events. Spontaneously occurring events at both loci will be analysed and compared. Using this approach, it will be possible to determine not only the salient mechanisms that occur in vivo, but the accuracy of the in vitro model. It will also be possible to examine in vitro the effects of agents commonly used to treat tumors, in the hope that treatments especially likely to lead to the expression of recessive oncogenes can be identified. Such treatments may be particularly harmful to individuals that carry a hereditary predisposition to malignancy.